The many flavoproteins that catalyze oxidation of carbon-nitrogen and carbon-oxygen bonds by transferring a hydride equivalent to the flavin play critical roles throughout metabolism. A number of these enzymes are targets for treatment of major diseases, so that better understanding of their mechanisms could provide insight for development of therapeutic agents. These enzymes also belong to multiple structural families; it is unclear if this reflects multiple strategies to catalyze essentially identical reactions or is a example of convergent evolution. We have interpreted previous studies of alcohol, keto acid, and amino acid oxidizing enzymes as consistent with a common hydride transfer mechanism for all, with CN and CO bond oxidizing enzymes differing in the need for an active site base in the latter. We now propose to carry out experiments to determine if enzymes that oxidize simple amines and polyamines utilize this same mechanism despite differences in protein and substrate structure. We will determine the mechanism of L- hydroxy-nicotine oxidase, an enzyme in the monoamine oxidase structural family that is proposed to catalyze the oxidation of a carbon-carbon bond rather than a carbon-nitrogen bond. We will continue our studies of polyamine oxidases, enzymes that oxidize the same substrates with different substrate specificities. 15N and 13C kinetic isotope effects will be used to determine the mechanism of amine oxidation, and analysis of the effects of site-directed mutations combined with crystallography will be used to determine the structural basis for the different specificities. We will measure 13C isotope effects for two structural classes of flavin amine oxidases to better define their transition state structures and to provide baseline values for other structural familis. We will determine if members of the trimethylamine dehydrogenase structural family use the same mechanism for amine oxidation as other structural families of amine-oxidizing enzymes. The results of these experiments will test our hypothesis that the different structural families of amine oxidizing flavoenzymes are examples of convergent evolution on a common catalytic mechanism, potentially providing a unifying mechanism for a structurally divergent group of enzymes. Finally, we will initiate studies of the oxidative reactions of flavin-dependent amine oxidases, to probe for intermediates in the reaction and for oxygen-binding sites.